Compositions and methods for treating alzheimers disease

ABSTRACT

Pharmaceutical compositions for treating Alzheimer&#39;s disease are disclosed. The pharmaceutical compositions include a compound having the general formula: 
     
       
         
         
             
             
         
       
     
     and a pharmaceutically acceptable carrier. Methods for treating Alzheimer&#39;s disease, inhibiting ATase I and/or ATase 2, reducing the activity of BACE1, reducing the level of amyloid β-peptide (Aβ), and/or reducing the level of APP intracellular domain peptide (AICD) by administering such compositions are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/492,146, filed on Jun. 1, 2011, which is incorporated by referenceherein.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under NS045669 andAG033514 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

The membrane protein β-site APP cleaving enzyme 1 (BACE1) is responsiblefor the β cleavage of the amyloid precursor protein (APP). The cleavage,which has been linked to the pathogenesis of Alzheimer's disease (AD),results in the generation of a small APP fragment (commonly referred toas C99) acting as the immediate substrate for γ secretase (Puglielli,2008). The sequential β/γ processing of APP results into two smallfragments, the amyloid β-peptide (Aβ) and the APP intracellular domain(AICD). Both have neurotoxic properties and both have been linked to thepathogenesis of AD (Cleary et al., 2005; Ghosal et al., 2009; Gilibertoet al., 2008; Haass and Steiner, 2001; Klein et al., 2001; Lambert etal., 1998; Lansbury, 1999; Puzzo et al., 2008). BACE1 acts as therate-limiting enzyme for these processing steps. As a result, geneticdisruption of BACE1 in the mouse abolishes both β and γ cleavage of APPand prevents AD neuropathology (Cai, 2001; Luo, 2001). Therefore,mechanisms that regulate levels and activity of BACE1 could be effectivetargets for the development of therapeutic agents.

We recently reported that nascent BACE1 is transiently acetylated onseven lysine residues in the lumen of the ER (Costantini, 2007) by twoER-based acetyl-CoA:lysine acetyltransferases which we named ATase1(also known as camello-like 2 and N-acetyltransferase 8B) and ATase2(also known as camello-like 1 and N-acetyltransferase 8) (Ko andPuglielli, 2009). The Nε-lysine acetylation regulates the ability ofnascent BACE1 to complete maturation. In fact, the acetylatedintermediates of the nascent protein are able to reach the Golgiapparatus and complete maturation while the non-acetylated intermediatesare retained and degraded in the ER Golgi intermediate compartment(ERGIC) (Costantini, 2007; Jonas, 2008). Ex vivo studies show that thelevels of BACE1 are tightly regulated by the ATases. In fact,up-regulation of ATase1 and ATase2 increases the levels of BACE1 and thegeneration of Aβ while siRNA-mediated down-regulation of eithertransferase achieves the opposite effects (Ko and Puglielli, 2009).

SUMMARY

The present inventors have identified biochemical inhibitors of ATase1and ATase2 that significantly reduce the levels of BACE1 and thegeneration of Aβ in cellular systems and in an animal model of AD. Theexperiments described herein confirm that the disclosed inhibitors canbe effectively used for the prevention and/or treatment of AD.

In a first aspect, the disclosure encompasses a pharmaceuticalcomposition that includes a pharmaceutically acceptable carrier and acompound of Formula I or Formula II, as set forth below.

In some embodiments, the composition comprises a compound selected fromcompounds of Formula I:

and pharmaceutically acceptable salts thereof. In some embodiments, R₁,R₂, R₃, R₄, and R₇ are each independently selected from hydrogen,halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₁₀ alkyl,wherein R₉ and R₁₀ are independently selected from C₁-C₅ alkyl. In someembodiments, R₈ is selected from hydrogen, halogen, —OH, —NH₂, and —SH,or is absent. In some embodiments, R₅ and R₆ are each independentlyselected from hydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉,—NR₉R₁₀, and C₁-C₁₀ alkyl, wherein R₉ and R₁₀ are independently selectedfrom C₁-C₅ alkyl. In some embodiments, R₅ and R₆ together are

wherein R₁₁, R₁₂, R₁₃, and R₁₄ are each independently selected fromhydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, andC₁-C₁₀ alkyl, wherein R₉ and R₁₀ are independently selected from C₁-C₅alkyl. In some embodiments, X₁ is selected from N, NH, O, and S. In someembodiments, X₂ is selected from N, O, and S. In some embodiments, X₃ isselected from —OH, ═O, —NH₂, ═NH, —SH, and ═S. In some embodiments, theX₁ to carbon a (C_(a)) bond and the carbon b (C_(b)) to carbon c (C_(c))bond are both double bonds, or the C_(a) to C_(b) bond is a double bond.

In some embodiments, R₁, R₃, R₄, and R₇ are each independently selectedfrom hydrogen and halogen. In some embodiments, R₁, R₃, and R₄ are eachhydrogen and R₇ is halogen. In some embodiments, R₅ and R₆ are eachindependently selected from hydrogen, halogen, and methyl. In someembodiments, R₅ and R₆ together are

wherein R₁₁, R₁₂, R₁₃, and R₁₄ are each independently selected fromhydrogen and halogen. In some embodiments, R₅ and R₆ are each hydrogenor halogen. In some embodiments, R₅ and R₆ together are

In some embodiments, R₂ is selected from hydrogen, halogen, —OH, —OR₉,—SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₆ alkyl, wherein R₉ and R₁₀ areindependently selected from C₁-C₃ alkyl. In some embodiments, R₈ isselected from hydrogen, halogen, —OH, and —SH, or is absent. In someembodiments, X₂ is N and R₈ is absent. In some embodiments, X₂ is O andR₈ is selected from hydrogen, halogen, —OH, and —SH.

In some embodiments, the compound is selected from the compounds inTable 1, and pharmaceutically acceptable salts thereof.

In some embodiments, a pharmaceutical composition comprises a compoundselected from compounds of Formula II:

and pharmaceutically acceptable salts thereof.

In some embodiments, R₁ is selected from hydrogen, halogen, —OH, —OR₁₁,—SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C₁₀ alkyl, wherein R₁₁ andR₁₂ are independently selected from C₁-C₆ alkyl. In some embodiments, R₂is selected from hydrogen, halogen, —OH, —SH, and —NH3. In someembodiments, R₃ to R₁₀ are each independently selected from hydrogen,halogen, —OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C₁₀alkyl, wherein R₁₁ and R₁₂ are independently selected from C₁-C₆ alkyl.In some embodiments, X₁ is selected from O, S, and NH. In someembodiments, X₂ and X₃ are each independently selected from O, S, andNH.

In some embodiments, R₃ to R₁₀ are each independently selected fromhydrogen, halogen, —OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, andC₁-C₁₀ alkyl, wherein R₁₁ and R₁₂ are independently selected from C₁-C₃alkyl. In some embodiments, R₃ to R₁₀ are each independently selectedfrom hydrogen and halogen. In some embodiments, R₁ is selected fromhydrogen, halogen, —OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, andC₁-C₁₀ alkyl, wherein R₁₁ and R₁₂ are independently selected from C₁-C₄alkyl. In some embodiments, R₁ is selected from hydrogen, halogen, —OH,—OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C₁₀ alkyl, wherein R₁₁and R₁₂ are independently selected from C₁-C₃ alkyl. In someembodiments, R₂ is selected from hydrogen and halogen. In someembodiments, X₁ is NH. In some embodiments, X₂ and X₃ are each O.

In some embodiments, the compound is selected from the compounds inTable 2, and pharmaceutically acceptable salts thereof.

In a second aspect, the disclosure encompasses methods of treating,slowing the progression of, delaying the onset of, or alleviating atleast one symptom of Alzheimer's disease. In some embodiments, the atleast one symptom is selected from memory loss, confusion, impairedjudgment, disorientation, and loss of language skills. In someembodiments, the method comprises administering to a patient apharmaceutical composition comprising an inhibitor of ATase1 and/orATase2. In some embodiments, the method comprises administering to apatient a pharmaceutical composition comprising a compound selected fromthe compounds of Formula I and II, and pharmaceutically acceptable saltsthereof.

In a third aspect, the disclosure encompasses methods of inhibiting atleast one acetyltransferase selected from ATase 1 and ATase 2 in a cell,reducing the activity of β-site APP cleaving enzyme 1 (BACE1) in a cell,or reducing the level of APP intracellular domain peptide (AICD) in acell. In some embodiments, the method comprises contacting the cell witha pharmaceutical composition comprising a compound selected from thecompounds of Formula I and II, including without limitation the specificcompounds disclosed in Table 1 and Table 2 below, and pharmaceuticallyacceptable salts thereof. In some embodiments, the cell is in vitro. Insome embodiments, the cell is in vivo.

In a fourth aspect, the disclosure encompasses the compounds of FormulaI and Formula II, including without limitation the specific compoundsdisclosed in Table 1 and Table 2 below, for use in treating Alzheimer'sdisease.

In a fifth aspect, the disclosure encompasses the compounds of Formula Iand Formula III, including without limitation the specific compoundsdisclosed in Table 1 and Table 2 below, for use in the manufacture of amedicament for treating Alzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. ATase1 and ATase2 are expressed in neurons and are up-regulatedin the brain of AD patients. (A) Western blot assessment of CHO cellsover-expressing human ATase1 and/or ATase2. The left panel was generatedwith an anti-ATase1/ATase2 antibody while the right panel was generatedwith an anti-myc antibody. Lane 1, control (untransfected) cells; lane2, stable over-expression of ATase1; lane 3, stable over-expression ofATase2; lane 4, stable over-expression of both ATase1 and ATase2. (B)Western blot showing the endogenous levels of ATase1 and ATase2 indifferent cellular systems. The right panel shows higher exposure of thesame image. Lane 1, CHO cells; lane 2, H4 cells; lane 3, SH-SY5Y cells;lane 4, SHEP cells; lane 5, PC-12 cells; lane 6, primary neurons (day 3in culture); lane 7, primary neurons (day 18 in culture); lane 8; wholebrain extract (cortex). Asterisks (*) indicates a background band thatmigrates immediately above the 28-kDa marker. (C) cDNA produced frombrain tissue (frontal cortex) of late-onset AD patients (n=5; averageage: 87; age range: 85-93) and age-matched controls (n=5; average age:88; age range: 86-91) was analyzed by quantitative real-time PCR.Results were normalized against GAPDH and are expressed as percent ofage-matched controls ±s.e.m. **, P<0.005.

FIG. 2. Inhibition kinetics of compound 9 and 19. (A) Schematicrepresentation of the chemical and structural features of compound 9 and19. (B and C) Lineweaver-Burke plots for compound 9 (B) and 19 (C).Results are the average of at least 6 independent determinations ±S.D.

FIG. 3. Compound 9 and 19 decrease the endogenous levels of BACE1. (A-F)H4 cells were treated with either compound 9 or 19 for 48 hours prior toWestern blot assessment of BACE1 levels in total cell lysates (A-D) andELISA determination of secreted Aβ (E). Representative Western blots areshown in (A) and (C) while respective quantifications are shown in (B)and (D). Results in (B), (D) and (E) are the average (n=6)±S.D. **,P<0.005.

FIG. 4. Compound 9 causes degradation of the ATases. (A) H4 cells weretreated with the indicated compounds (10 μM) for 48 hours prior toWestern blot assessment of BACE1 and ATase1/ATase2 levels in total celllysates. (B, D and E) Quantification of changes are expressed as percentof control (no treatment) and are the average (n=3)±S.D. *, P<0.05; **,P<0.005; ^(#), P<0.0005. (C) ELISA determination of secreted Aβ in theconditioned media of the experiment described in (A). Results areexpressed as percent of control (no treatment) and are the average(n=3)±S.D. *, P<0.05; **, P<0.005. (F) CHO cells over-expressing eitherATase1 or ATase2 were treated with compound 9 (10 μM) for 48 hours priorto Western blot assessment of ATase1 and ATase2 levels in total celllysates.

FIG. 5. ATase2 is more tightly regulated by lipid second messengerceramide. H4 cells were treated with 10 μM ceramide for the indicatedtime prior to quantitative real-time PCR. Results are the average(n=4)±S.D. *, P<0.05.

FIG. 6. Summary of the strategy used to identify the compounds tested inthis study. Of the 186 compounds identified with the in vitro assay, 93were unique to our screen (never identified as positive hits in otherscreens). The cytotoxicity assay included Calcein AM incorporation,Cell-Titer glo and Mitotracker Red.

FIG. 7. Compound 9 and 19 decrease levels and activity of BACE1. H4cells were treated with either compound 9 or 19 for 48 hours prior toWestern blot assessment of BACE1 and C99 levels in cell lysates. C99 isthe immediate product of BACE1-mediated cleavage of APP.

FIG. 8. Compound 9 and 19 do not affect the lysine acetylation profileof cytosolic or nuclear proteins. (A and B) H4 cells were treated witheither compound 9 or 19 for 48 hours prior to Western blot assessment ofthe acetylation profile of cytosolic (A) and nuclear (B) proteins. (C)The nuclear fraction used in (B) was also used to assess the lysineacetylation profile of the histone proteins H3 and H4.

FIG. 9. Schematic representation of the chemical and structural featuresof compound 9.E, 9.F and 9.G. The site of relevant modification in theparent compound is circled. The structure is shown as pseudo-top andfront views.

FIG. 10. Schematic representation of the chemical features of compound9.A, 9.B, 9.0 and 9.D. The sites of relevant modification in the parentcompound are circled.

FIG. 11. Schematic representation of the chemical features of compound9.H-9.M.

FIG. 12. Schematic representation of the chemical features of compound19.A, 19.B and 19.C. The site of relevant modification in the parentcompound is circled.

FIG. 13. Compound 9 decreases the levels of both ATase1 and ATase2. (A)H4 cells were treated with the increasing concentrations of compound 9for 48 hours prior to Western blot assessment of BACE1 and ATase1/ATase2levels in total cell lysates. (B-D) Quantification of changes areexpressed as percent of control (no treatment) and are the average(n=3)±S.D. *, P<0.05; **, P<0.005; ^(#), P<0.0005.

FIG. 14. Compound 9 is able to cross the BBB. Wild-type mice (n=5) werefed a diet containing 50 mg/kg/day of compound 9 for one week and thensacrificed. Control animals (n=5) received placebo-containing pellets.CSF was collected and analyzed by mass spectrometry (MS) to detectcompound 9. The pick corresponding to compound 9 is indicated. Resultsshow MS data for 2 treated (center and rightmost mass spectra) and 1control (placebo) animal (leftmost mass spectrum). Because of the stronghydrophobic properties of the compound, elution was in 100% methanol.Under these conditions most of compound 9 was found protonated andcomplexed as follows: 1 molecule of compound 9 (protonated):3 moleculesof water (1 protonated):1 molecule of methanol. The total mass of theeluted complex was 369.4. This complex is only generated when addingmethanol during the elution process for the MS. Compound 9 was neverobserved in control animals.

FIG. 15. Compound 9 prevents synaptic deficits in APP_(695/swe) mice.Non-Transgenic (Non-Tg) and APP_(695/swe) mice were fed a dietcontaining 50 mg/kg/day of compound 9 for 4 months prior to synapticassessment. Compound 9 prevented the severe deficits of the latecomponent of long term potentiation (LTP) that characterizes theAPP_(695/swe) phenotype (A). The compound did not affect the intrinsicproperties of synaptic activity in Non-Tg animals (B). Field excitatorypostsynaptic potential (fEPSP) as percent of baseline (100) is shown asa function of time. Calibration: 1 mV, 1 ms. All values are mean±SEM.*P<0.0001.

FIG. 16. Compound 9 decreases levels of BACE1 in brain tissuehomogenates of APP_(695/swe) transgenic mice. Compound 9 wasadministered to select transgenic mice for four months, beginning atweaning, with additional transgenic mice used as controls. Brain tissuehomogenates were obtained from sacrificed transgenic mice and Westernblot assessment of BACE1 in brain tissue homogenates was performed.

DETAILED DESCRIPTION

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Certain techniques used in connection with recombinant DNA,oligonucleotide synthesis, tissue culture and transformation (e.g.,electroporation, lipofection), enzymatic reactions, and purificationtechniques are known in the art. Many such techniques and procedures aredescribed, e.g., in Sambrook et al. Molecular Cloning: A LaboratoryManual (2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)), among other places. In addition, certaintechniques for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients arealso known in the art.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

DEFINITIONS

In this application, the use of “or” means “and/or” unless statedotherwise. In the context of a multiple dependent claim, the use of “or”refers back to more than one preceding independent or dependent claim inthe alternative only. Also, terms such as “element” or “component”encompass both elements and components comprising one unit and elementsand components that comprise more than one subunit unless specificallystated otherwise.

As used herein, “about,” “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” or“approximately” will mean up to plus or minus 10% of the particular termand “substantially” and “significantly” will mean more than plus orminus 10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.”

As used herein, the term “inhibitor of ATase1 and/or ATase2” refers to amolecule that interacts with ATase1 and/or ATase2 and reduces theactivity of ATase1 and/or ATase2 by at least 30% according to an assaydescribed herein. In some embodiments, an inhibitor of ATase1 and/orATase2 reduces the activity of ATase1 and/or ATase2 by at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Insome embodiments, an inhibitor of ATase1 and/or ATase2 is a smallmolecule. In some embodiments, an inhibitor of ATase1 and/or ATase2 is acompound selected from the compounds of Formula I and Formula II, andpharmaceutically acceptable salts thereof. Inhibitors of ATase1 and/orATase2 include inhibitors of ATase1, inhibitors of ATase2, andinhibitors of ATase1 and ATase 2.

As used herein, the term “inhibitor of ATase1” refers to a molecule thatinteracts with ATase1 and reduces the activity of ATase1 by at least 30%according to an assay described herein. In some embodiments, aninhibitor of ATase1 reduces the activity of ATase1 by at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Insome embodiments, an inhibitor of ATase1 is a small molecule. In someembodiments, an inhibitor of ATase1 is a compound selected from thecompounds of Formula I and Formula II, and pharmaceutically acceptablesalts thereof.

As used herein, the term “inhibitor of ATase2” refers to a molecule thatinteracts with ATase2 and reduces the activity of ATase2 by at least 30%according to an assay described herein. In some embodiments, aninhibitor of ATase2 reduces the activity of ATase2 by at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Insome embodiments, an inhibitor of ATase2 is a small molecule. In someembodiments, an inhibitor of ATase2 is a compound selected from thecompounds of Formula I and Formula II, and pharmaceutically acceptablesalts thereof.

As used herein, the term “inhibitor of ATase1 and ATase2” refers to amolecule that interacts with both ATase1 and ATase2 and reduces theactivity of each of ATase1 and ATase2 by at least 30% according to anassay described herein. In some embodiments, an inhibitor of ATase1 andATase2 reduces the activity of each of ATase1 and ATase2 by at least40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least90% (although it may not reduce the activity of ATase1 and ATase2 to thesame extent). In some embodiments, an inhibitor of ATase1 and ATase2 isa small molecule. In some embodiments, an inhibitor of ATase1 and ATase2is a compound selected from the compounds of Formula I and Formula II,and pharmaceutically acceptable salts thereof.

The terms “patient” and “subject” are used interchangeably to refer to arefer to one who receives medical care, attention or treatment. As usedherein, the term is meant to encompass a person diagnosed with a diseasesuch as a Alzheimer's disease or at risk for developing Alzheimer'sdisease. A “patient in need thereof” may include a patient having,suspected of having, or at risk for developing Alzheimer's disease orsymptoms thereof.

As used herein, the term “treatment,” “treating,” or “treat” refers tocare by procedures or application that are intended to alleviate adisease or symptoms of a disease (including reducing the occurrence ofsymptoms of the disease). Although it is preferred that treating acondition or disease such as Alzheimer's disease will result in animprovement of the condition or disease, the term treating as usedherein does not indicate, imply, or require that the procedures orapplications are always successful in treating the condition disease oralleviating symptoms associated with the condition or disease. Treatinga patient may result in adverse side effects or, in some instances, evena worsening of the condition which the treatment was intended toimprove. Treating may include treating a patient having, suspected ofhaving, or at risk for developing Alzheimer's disease or symptomsthereof. In some embodiments, treating Alzheimer's disease results in areduction in the amount of Aβ peptide in the brain and/or a reduction inthe number and/or size of amyloid plaques.

As used herein, the term “prevention,” “preventing,” or “prevent” refersto care by procedures or application that are intended to inhibit orstop development of a disease or symptoms of a disease (includinginhibiting or stopping the occurrence of symptoms of the disease).Although it is preferred that preventing a condition or disease such asAlzheimer's disease will result in a failure of the condition or diseaseto develop, the term preventing as used herein does not indicate, imply,or require that the procedures or applications are always successful ininhibiting development of the condition or disease or symptomsassociated with the condition or disease. Preventing a condition ordisease may result in adverse side effects or, in some instances, even aworsening of the condition or disease that the treatment was intended toprevent. Methods of preventing a condition or disease may includetreating a patient suspected of having, or at risk for developingAlzheimer's disease or symptoms thereof. In some embodiments, preventingAlzheimer's disease means that elevated levels of Aβ peptide are notformed and/or amyloid plaques are not formed in the brain.

As used herein, the term “slowing the progression of” or “slows theprogression of” refers to care by procedures or application that areintended to reduce the rate of development of a disease or symptoms of adisease (including reducing the rate of occurrence of symptoms of thedisease). Although it is preferred that slowing the progression of acondition or disease such as Alzheimer's disease will result in areduction in the rate that the condition or disease develops, the term“slowing the progression of” as used herein does not indicate, imply, orrequire that the procedures or applications are always successful inslowing the rate of development of the condition or disease or symptomsassociated with the condition or disease. Slowing the progression of acondition or disease may result in adverse side effects or, in someinstances, even a worsening of the condition or disease that thetreatment was intended to slow. Methods of slowing the progression of acondition or disease may include treating a patient having, suspected ofhaving, or at risk for developing Alzheimer's disease or symptomsthereof. In some embodiments, slowing the progression of Alzheimer'sdisease results in a reduction in the rate of formation of Aβ peptide inthe brain and/or a reduction in rate of formation of amyloid plaques(the number and/or size of amyloid plaques).

As used herein, the term “delaying the onset of” or “delays the onsetof” refers to care by procedures or application that are intended todelay in time the development of a disease or symptoms of a disease(including delaying in time the appearance or occurrence of symptoms ofthe disease). Although it is preferred that delaying the onset of acondition or disease such as Alzheimer's disease will result in a delayin time before the condition or disease develops, the term “delaying theonset of” as used herein does not indicate, imply, or require that theprocedures or applications are always successful in delaying the onsetof the condition or disease or symptoms associated with the condition ordisease. Delaying the onset of a condition or disease may result inadverse side effects or, in some instances, even a worsening of thecondition or disease that the treatment was intended to delay. Methodsof delaying the onset of a condition or disease may include treating apatient suspected of having, or at risk for developing Alzheimer'sdisease or symptoms thereof. In some embodiments, delaying the onset ofAlzheimer's disease results in a reduction in the rate of formation ofAβ peptide in the brain and/or a reduction in rate of formation ofamyloid plaques (the number and/or size of amyloid plaques).

As used herein the term “effective amount” refers to the amount or doseof the agent, upon single or multiple dose administration to thesubject, which provides the desired effect in the subject underdiagnosis or treatment. The disclosed methods may include administeringan effective amount of the disclosed compounds (e.g., as present in apharmaceutical composition) for treating Alzheimer's disease in thepatient, whereby the effective amount alleviates a molecular cause orsymptom of Alzheimer's disease (including reducing the occurrence ofsuch causes or symptoms of Alzheimer's disease).

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount or dose of agent administered, a numberof factors can be considered by the attending diagnostician, such as:the species of the patient; its size, age, and general health; theparticular symptoms or the severity of the disease; the response of theindividual patient; the particular agent administered; the mode ofadministration; the bioavailability characteristics of the preparationadministered; the dose regimen selected; the use of concomitantmedication; and other relevant circumstances.

The phrase “alleviates at least one symptom,” as used herein, means thata particular treatment results in a lessening of at least one symptom ofa disease. Such lessening of a symptom may be a qualitative orquantitative reduction in the severity of the symptom, or may be areduction in the number of occurrences of the symptom, even though eachoccurrence may be as severe as it was before the treatment (one or moreoccurrences may also be less severe). Nonlimiting exemplary symptoms ofAlzheimer's disease include memory loss, confusion, impaired judgment,disorientation, and loss of language skills.

Exemplary Methods

In some embodiments, methods of treating Alzheimer's disease areprovided. In some embodiments, methods of alleviating at least onesymptom thereof are provided. In some embodiments, the at least onesymptom is selected from memory loss, confusion, impaired judgment,disorientation, and loss of language skills. In some embodiments,methods of preventing Alzheimer's disease are provided. In someembodiments, methods of slowing the progression of Alzheimer's diseaseare provided. In some embodiments, methods of delaying the onset ofAlzheimer's disease are provided.

In some embodiments, a method comprises administering to a patient aninhibitor of ATase1 and/or ATase2. In some embodiments, a methodcomprises administering to a patient a compound selected from thecompounds of Formula I and pharmaceutically acceptable salts thereofand/or a compound selected from the compounds of Formula II andpharmaceutically acceptable salts thereof. In some embodiments, a methodcomprises administering to a patient a compound selected from thecompounds in Tables 1 and 2 below and pharmaceutically acceptable saltsthereof. In some embodiments, the compound is comprised in apharmaceutical composition suitable for administration to a patient.

Techniques for formulation and administration can be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.The mode of administration can be selected to maximize delivery to adesired target site in the body. Suitable routes of administration can,for example, include oral, rectal, transmucosal, transcutaneous, orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. Further, a pharmaceutical composition can bedelivered locally, for example, by direct injection into a specifictissue.

For any compounds of the present invention, the therapeuticallyeffective dose can be estimated initially from in vitro assays or usingart-recognized animal model systems or a combination thereof. Forexample, a dose can be formulated in animal models to achieve acirculating concentration range that includes the EC50 (effective dosefor 50% decrease) as determined in vitro, i.e., the concentration of thetest compound which achieves a half-maximal reduction in ATase1 and/orATase 2 activity. Such information can be used to more accuratelydetermine useful doses in humans.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the compounds employed, body weight, general health, sex,diet, time of administration, route of administration, and rate ofexcretion, and other drugs currently taken by the patient.

In some embodiments, methods of inhibiting at least oneacetyltransferases selected from ATase 1 and ATase 2 in a cell areprovided. In some embodiments, methods of reducing the activity ofβ-site APP cleaving enzyme 1 (BACE1) in a cell are provided. In someembodiments, methods of reducing the level of amyloid β-peptide (Aβ) ina cell are provided. In some embodiments, methods reducing the level ofAPP intracellular domain peptide (AICD) in a cell are provided.

In some embodiments, such methods comprise contacting the cell with acompound selected from the compounds of Formula I and pharmaceuticallyacceptable salts thereof and/or a compound selected from the compoundsof Formula II and pharmaceutically acceptable salts thereof. In someembodiments, the compound is selected from the compounds in Table 1 andTable 2, and pharmaceutically acceptable salts thereof. The cell may bein vitro or may be in vivo (i.e., may be comprised within an organism,such as a human patient). In some embodiments in which the cell iscomprised in a patient, the method comprises administering the compoundto the patient. Cells in vitro may be in the form of dissociated cells,cells in suspension culture, cells in monolayer culture, cells in 3Dculture, cells comprised in ex vivo tissues, cells comprised in ex vivoorgans, and the like.

Exemplary Compounds and Pharmaceutical Compositions

In some embodiments, compounds of Formula I and pharmaceuticallyacceptable salts thereof are provided:

In some embodiments, R₁, R₂, R₃, R₄, and R₇ are each independentlyselected from hydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉,—NR₉R₁₀, and C₁-C₁₀ alkyl. In some embodiments, R₁, R₂, R₃, R₄, and R₇are each independently selected from hydrogen, halogen, —OH, —OR₉, —SH,—SR₉, —NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₈ alkyl. In some embodiments, R₁,R₂, R₃, R₄, and R₇ are each independently selected from hydrogen,halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₆ alkyl. Insome embodiments, R₁, R₂, R₃, R₄, and R₇ are each independently selectedfrom hydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, andC₁-C₆ alkyl. In some embodiments, R₉ and R₁₀ are independently selectedfrom C₁-C₅ alkyl. In some embodiments, R₉ and R₁₀ are independentlyselected from C₁-C₄ alkyl. In some embodiments, R₉ and R₁₀ areindependently selected from C₁-C₃ alkyl. In some embodiments, R₉ and R₁₀are independently selected from C₁-C₂ alkyl. In some embodiments, R₁,R₃, R₄, and R₇ are each independently selected from hydrogen andhalogen. In some embodiments, R₁, R₃, and R₄ are each hydrogen and R₇ ishalogen. In some embodiments, R₁, R₃, R₄, and R₇ are hydrogen.

In some embodiments, R₂ is selected from hydrogen, halogen, —OH, —OR₉,—SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₆ alkyl. In some suchembodiments, R₉ and R₁₀ are independently selected from C₁-C₃ alkyl. Insome such embodiments, R₁, R₃, R₄, and R₇ are each independentlyselected from hydrogen and halogen. In some such embodiments, R₁, R₃,and R₄ are hydrogen, and R₇ is halogen.

In some embodiments, R₈ is selected from hydrogen, halogen, —OH, —NH₂,and —SH. In some embodiments, R₈ is selected from hydrogen, halogen,—OH, and —SH. In some embodiments, for example when the bond connectingX₂ to the carbon shared by X₂ and R₈ is a double bond, R₈ is absent.

In some embodiments, R₅ and R₆ are each independently selected fromhydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, andC₁-C₁₀ alkyl. In some embodiments, R₅ and R₆ are each independentlyselected from hydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉,—NR₉R₁₀, and C₁-C₈ alkyl. In some embodiments, R₅ and R₆ are eachindependently selected from hydrogen, halogen, —OH, —OR₉, —SH, —SR₉,—NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₆ alkyl. In some embodiments, R₅ and R₆are each independently selected from hydrogen, halogen, —OH, —OR₉, —SH,—SR₉, —NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₄ alkyl. In some embodiments, R₉ andR₁₀ are independently selected from C₁-C₅ alkyl. In some embodiments, R₉and R₁₀ are independently selected from C₁-C₄ alkyl. In someembodiments, R₉ and R₁₀ are independently selected from C₁-C₃ alkyl. Insome embodiments, R₉ and R₁₀ are independently selected from C₁-C₂alkyl. It is to be understood that each R₉ and R₁₀ is independentlyselected for each R₁, R₂, R₃, R₄, R₅, R₆, R₇, etc., that comprises an R₉and/or an R₁₀. Thus, for example, in some embodiments, an R₉ on an R₁substituent need not be the same as an R₉ on an R₂ substituent in thesame compound.

In some embodiments, R₅ and R₆ together are

In some such embodiments, R₁₁, R₁₂, R₁₃, and R₁₄ are each independentlyselected from hydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉,—NR₉R₁₀, and C₁-C₁₀ alkyl. In some such embodiments, R₁₁, R₁₂, R₁₃, andR₁₄ are each independently selected from hydrogen, halogen, —OH, —OR₉,—SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₈ alkyl. In some suchembodiments, R₁₁, R₁₂, R₁₃, and R₁₄ are each independently selected fromhydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₆alkyl. In some such embodiments, R₁₁, R₁₂, R₁₃, and R₁₄ are eachindependently selected from hydrogen, halogen, —OH, —OR₉, —SH, —SR₉,—NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₄ alkyl. In some embodiments, R₉ and R₁₀are independently selected from C₁-C₅ alkyl. In some embodiments, R₉ andR₁₀ are independently selected from C₁-C₃ alkyl. It is to be understoodthat each R₉ and R₁₀ is independently selected for each substituent thatcomprises an R₉ and/or R₁₀. In some such embodiments, R₁₁, R₁₂, R₁₃, andR₁₄ are each independently selected from hydrogen and halogen. In someembodiments, R₁₁, R₁₂, R₁₃, and R₁₄ are hydrogen.

In some embodiments, R₅ and R₆ are each independently selected fromhydrogen, halogen, and methyl. In some embodiments, R₅ and R₆ togetherare

In some embodiments, X₁ is selected from N, NH, O, and S. That is, insome embodiments, when X₁ is N, the X₁ to carbon a (C_(a)) bond is adouble bond. In some such embodiments, the carbon b (C_(b)) to carbon c(C_(c)) bond is also a double bond. In some embodiments, when X₁ is NH,O, or S, the C_(a) to C_(b) bond is a double bond.

In some embodiments, X₂ is selected from N, O, and S. In someembodiments, when X₂ is N, R₈ is absent, and the bond between X₂ and thecarbon to which R₈ is shown attached in Formula I is a double bond. Insome embodiments, X₂ is O and R₈ is selected from hydrogen, halogen,—OH, and —SH.

In some embodiments, X₃ is selected from —OH, ═O, —NH₂, ═NH, —SH, and═S.

In some embodiments, a compound of Formula I is provided, wherein X₁ isO; X₂ is N; X₃ is selected from ═O, ═S, and ═NH; the carbon a (C_(a)) tocarbon b (C_(b)) bond is a double bond; R₈ is absent; R₁, R₃, R₄, and R₇are each independently selected from H and halogen; R₂ is selected fromhydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH2, —NHR₉, —NR₉R₁₀, and C₁ toC₆ alkyl, wherein R₉ and R₁₀ are each independently selected from C₁ toC₄ alkyl; and R₅ and R₆ are together are

wherein R₁₁, R₁₂, R₁₃, and R₁₄ are each independently selected fromhydrogen and halogen. In some such embodiments, R₂ is selected fromhydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH2, —NHR₉, —NR₉R₁₀, and C₁ toC₄ alkyl, wherein R₉ and R₁₀ are each independently selected from C₁ toC₃ alkyl.

In some embodiments, a compound of Formula I is provided, wherein X₁ isN; X₂ is N; X₃ is selected from —OH, —SH, and —NH₂; the X₁ to carbon a(C_(a)) bond is a double bond and the carbon b (C_(b)) to carbon c(C_(c)) bond is a double bond; R₈ is absent; R₁, R₃, R₄, and R₇ are eachindependently selected from H and halogen; R₂ is selected from hydrogen,halogen, —OH, —OR₉, —SH, —SR₉, —NH2, —NHR₉, —NR₉R₁₀, and C₁ to C₆ alkyl,wherein R₉ and R₁₀ are each independently selected from C₁ to C₄ alkyl;and R₅ and R₆ are together are

wherein R₁₁, R₁₂, R₁₃, and R₁₄ are each independently selected fromhydrogen and halogen. In some such embodiments, R₂ is selected fromhydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH2, —NHR₉, —NR₉R₁₀, and C₁ toC₄ alkyl, wherein R₉ and R₁₀ are each independently selected from C₁ toC₃ alkyl. In some such embodiments, R₂ is selected from hydrogen andhalogen.

In some embodiments, a compound of Formula I is provided, wherein X₁ isNH; X₂ is O; X₃ is selected from ═O, ═S, and ═NH; the carbon a (C_(a))to carbon b (C_(b)) bond is a double bond; R₈ is selected from hydrogen,halogen, —OH, —SH, and —NH₂; R₁, R₃, R₄, and R₇ are each independentlyselected from H and halogen; R₂ is selected from hydrogen, halogen, —OH,—OR₉, —SH, —SR₉, —NH2, —NHR₉, —NR₉R₁₀, and C₁ to C₆ alkyl, wherein R₉and R₁₀ are each independently selected from C₁ to C₄ alkyl; and R₅ andR₆ are together are

wherein R₁₁, R₁₂, R₁₃, and R₁₄ are each independently selected fromhydrogen and halogen. In some such embodiments, R₂ is selected fromhydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH2, —NHR₉, —NR₉R₁₀, and C₁ toC₄ alkyl, wherein R₉ and R₁₀ are each independently selected from C₁ toC₃ alkyl. In some such embodiments, R₂ is selected from hydrogen andhalogen.

In some embodiments, a compound of Formula I is provided, wherein X₁ isO; X₂ is N; X₃ is selected from ═O, ═S, and ═NH; the carbon a (C_(a)) tocarbon b (C_(b)) bond is a double bond; R₈ is absent; R₁, R₃, R₄, R₅,R₆, and R₇ are each independently selected from H and halogen; and R₂ isselected from hydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH2, —NHR₉,—NR₉R₁₀, and C₁ to C₆ alkyl, wherein R₉ and R₁₀ are each independentlyselected from C₁ to C₄ alkyl. In some such embodiments, R₂ is selectedfrom hydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH2, —NHR₉, —NR₉R₁₀, andC₁ to C₄ alkyl, wherein R₉ and R₁₀ are each independently selected fromC₁ to C₃ alkyl. In some such embodiments, R₂ is selected from hydrogen,halogen, —OH, —SH, and —NH₂.

In some embodiments, a compound of Formula I is selected from thecompounds in Table 1, and pharmaceutically acceptable salts thereof

TABLE 1 Nonlimiting exemplary compounds of Formula I Compound Structure9

9A

9B

9C

9D

9E

9I

In some embodiments, compounds of Formula II and pharmaceuticallyacceptable salts thereof are provided:

In some embodiments, R₁ is selected from hydrogen, halogen, —OH, —OR₁₁,—SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C₁₀ alkyl. In someembodiments, R₁ is selected from hydrogen, halogen, —OH, —OR₁₁, —SH,—SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C₈ alkyl. w In some embodiments,R₁ is selected from hydrogen, halogen, —OH, —OR₁₁, —SH, —SR₁₁, —NH₂,—NHR₁₁, —NR₁₁R₁₂, and C₁-C₆ alkyl. In some embodiments, R₁ is selectedfrom hydrogen, halogen, —OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂,and C₁-C₄ alkyl. In some embodiments, R₁₁ and R₁₂ are independentlyselected from C₁-C₆ alkyl. In some embodiments, R₁₁ and R₁₂ areindependently selected from C₁-C₅ alkyl. In some embodiments, R₁₁ andR₁₂ are independently selected from C₁-C₄ alkyl. In some embodiments,R₁₁ and R₁₂ are independently selected from C₁-C₃ alkyl. In someembodiments, R₁₁ and R₁₂ are independently selected from C₁-C₂ alkyl. Itis to be understood that each R₁₁ and R₁₂ is independently selected foreach substituent that comprises an R₁₁ and/or R₁₂.

In some embodiments, R₂ is selected from hydrogen, halogen, —OH, —SH,and —NH₂. In some embodiments, R₂ is selected from hydrogen and halogen.

In some embodiments, R₃ to R₁₀ are each independently selected fromhydrogen, halogen, —OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, andC₁-C₁₀ alkyl. In some embodiments, R₃ to R₁₀ are each independentlyselected from hydrogen, halogen, —OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁,—NR₁₁R₁₂, and C₁-C₈ alkyl. In some embodiments, R₃ to R₁₀ are eachindependently selected from hydrogen, halogen, —OH, —OR₁₁, —SH, —SR₁₁,—NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C₆ alkyl. In some embodiments, R₃ to R₁₀are each independently selected from hydrogen, halogen, —OH, —OR₁₁, —SH,—SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C4 alkyl. In some embodiments, R₁₁and R₁₂ are independently selected from C₁-C₆ alkyl. In someembodiments, R₁₁ and R₁₂ are independently selected from C₁-C₅ alkyl. Insome embodiments, R₁₁ and R₁₂ are independently selected from C₁-C₄alkyl. In some embodiments, R₁₁ and R₁₂ are independently selected fromC₁-C₃ alkyl. It is to be understood that each R₁₁ and R₁₂ isindependently selected for each substituent that comprises an R₁₁ and/orR₁₂.

In some embodiments, R₃ to R₁₀ are each independently selected fromhydrogen and halogen.

In some embodiments, X₁ is selected from O, S, and NH. In someembodiments, X₁ is NH. In some embodiments, X₂ and X₃ are eachindependently selected from O, S, and NH. In some embodiments, X₂ and X₃are each O.

In some embodiments, a compound of Formula II is provided, wherein X₁ isselected from O, S, and NH; X₂ and X₃ are each independently selectedfrom ═O, ═S, and ═NH; R₂ to R₁₀ are each independently selected fromhydrogen and halogen; and R₁ is selected from hydrogen, halogen, —OH,—OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C₄ alkyl, wherein R₁₁and R₁₂ are independently selected from C₁-C₃ alkyl. In some suchembodiments, R₁ is selected from hydrogen, halogen, —OH, —OR₁₁, —SH,—SR₁₁, —NH₂, —NHR₁₁, and C₁-C₃ alkyl, wherein R₁₁ is selected from C₁-C₂alkyl. In some such embodiments, R₁ is selected from hydrogen, halogen,—OH, —OR₁₁, —SH, and —SR₁₁, wherein R₁₁ is selected from C₁-C₂ alkyl.

In some embodiments, a compound of Formula II is provided, wherein X₁ isNH; X₂ and X₃ are each independently selected from ═O and ═S; R₂ to R₁₀are each independently selected from hydrogen and halogen; and R₁ isselected from hydrogen, halogen, —OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁,—NR₁₁R₁₂, and C₁-C₄ alkyl, wherein R₁₁ and R₁₂ are independentlyselected from C₁-C₃ alkyl. In some such embodiments, R₁ is selected fromhydrogen, halogen, —OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁, and C₁-C₃alkyl, wherein R₁₁ is selected from C₁-C₂ alkyl. In some suchembodiments, R₁ is selected from hydrogen, halogen, —OH, —OR₁₁, —SH, and—SR₁₁, wherein R₁₁ is selected from C₁-C₂ alkyl.

In some embodiments, a compound of Formula II is provided, wherein X₁ isNH; X₂ and X₃ are each ═O; R₂ to R₁₀ are each independently selectedfrom hydrogen and halogen; and R₁ is selected from hydrogen, halogen,—OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C₄ alkyl, whereinR₁₁ and R₁₂ are independently selected from C₁-C₃ alkyl. In some suchembodiments, R₁ is selected from hydrogen, halogen, —OH, —OR₁₁, —SH,—SR₁₁, —NH₂, —NHR₁₁, and C₁-C₃ alkyl, wherein R₁₁ is selected from C₁-C₂alkyl. In some such embodiments, R₁ is selected from hydrogen, halogen,—OH, —OR₁₁, —SH, and —SR₁₁, wherein R₁₁ is selected from C₁-C₂ alkyl.

In some embodiments, a compound of Formula II is selected from thecompounds in Table 2, and pharmaceutically acceptable salts thereof

TABLE 2 Nonlimiting exemplary compounds of Formula II Compound Structure19

19A

19B

The term “alkyl,” as used herein, refers to straight- or branched-chainhydrocarbon radical, which may be fully saturated, or mono- orpoly-unsaturated, having the number of carbon atoms designated (i.e.,C₁-C₁₀ means one to ten carbons). Examples of saturated hydrocarbonradicals include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. An unsaturated alkylgroup is one having one or more double bonds or triple bonds. Examplesof unsaturated alkyl groups include, but are not limited to, vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,ethynyl, 1- and 3-propynyl, 3-butynyl, etc. The term “alkyl,” unlessotherwise noted, includes derivatives of alkyl, including heteroalkyl,substituted alkyl, and substituted heteroalkyl. Alkyl groups that arelimited to hydrocarbon groups may be referred to as “homoalkyl”.

A “substituted” alkyl or heteroalkyl, as used herein, refers to astraight- or branched-chain alkyl or heteroalkyl that comprises at leastone substituent comprising a non-carbon atom selected from halogen, O,N, S, and Se. Nonlimiting exemplary substituents include halogen, —OH,═O, —CO₂, —CO₂R, —NH₂, ═NH, —NHR, —NR′R″, ═N—R, —SH, —SR, —CN, —NO,—NO₂, etc., wherein R, R′, and R″ are independently selected from C₁-C₃homoalkyl.

A “heteroalkyl,” as used herein, refers to an straight- orbranched-chain alkyl in which one or more carbon atoms other than thecarbon atom that is attached to the molecule of Formula I or Formula IIis replaced with an atom selected from O, N, S, or Se (along with asuitable change in the number of attached atoms according to the valencyof the atom replacing carbon). Thus, a C₄ heteroalkyl comprises one ormore heteroatoms and three or fewer carbon atoms. Nonlimiting exemplaryC₄ heteroalkyls therefore include, but are not limited to,—CH₂—NH—CH₂—CH₃, —CHF—CH₂—O—CF₃, —CH(CH₃)—S—CH₃, etc. Certain groups maybe characterized in multiple ways. As a nonlimiting example,—CH₂—NH—CH₂—OH may be described as a C₄ heteroalkyl or a C₃ substitutedheteroalkyl. In such instances, the description that results in thegroup being included in a genus controls, unless indicated otherwise.

The term “halogen,” as used herein, refers to an atom selected from F,Cl, Br, and I.

Provided herein are pharmaceutical compositions. Such compositionsinclude a therapeutically effective amount of a compound of Formula Iand/or a compound of Formula II, and a pharmaceutically acceptablecarrier. As used herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, for example, in humans. Further, a“pharmaceutically acceptable carrier” will generally be a non-toxicsolid, semisolid or liquid filler, diluent, adjuvant, excipient,vehicle, encapsulating material, or formulation auxiliary of any type.The particular carrier or carriers included in a pharmaceuticalcomposition are typically dependent on the chosen route ofadministration.

Such pharmaceutically acceptable carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, including peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueoussolutions comprising dextrose, glycerol, sorbitol, sodium carboxymethylcellulose, and the like, can also be employed as liquid carriers,particularly for injectable solutions. In some embodiments, the activeingredient and one or more carriers are in dry form and can bereconstituted with a suitable vehicle, such as water, before use.

Suitable pharmaceutical carriers also include excipients such as starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain wetting oremulsifying agents, or pH buffering agents such as acetates, citrates,phosphates, Hank's solution, Ringer's solution, and lactated Ringer'ssolution; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. For transmucosaland transcutaneous administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Compositions can also be formulated for inhalation.Examples of suitable pharmaceutical carriers and formulations aredescribed, e.g., in “Remington's Pharmaceutical Sciences” by E. W.Martin. A nonlimiting exemplary pharmaceutical carrier for hydrophobicembodiments of the compounds of the present invention is a co-solventsystem comprising benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. A nonlimitingexemplary co-solvent system is the VPD co-solvent system. VPD is asolution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactantpolysorbate 80, and 65% w/v polyethylene glycol 300, made up to volumein absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPDdiluted 1:1 with a 5% dextrose in water solution. This co-solvent systemdissolves hydrophobic compounds well, and itself produces low toxicityupon systemic administration. The proportions of a co-solvent system canbe varied considerably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentscan be varied; for example, other low-toxicity nonpolar surfactants canbe used instead of polysorbate 80; the fraction size of polyethyleneglycol can be varied; other biocompatible polymers can replacepolyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars orpolysaccharides can substitute for dextrose.

Alternatively, liposomes and emulsions are well known examples ofdelivery vehicles or carriers for hydrophobic drugs. Certain organicsolvents such as dimethylsulfoxide also can be employed, althoughusually at the cost of greater toxicity. Additionally, compounds of thepresent invention can be delivered using a sustained-release system,such as semipermeable matrices of solid hydrophobic polymers containingthe therapeutic agent. Various sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules can, depending on their chemical nature,release a drug for a few weeks up to over 100 days.

Pharmaceutical compositions described herein generally contain atherapeutically effective amount of one or more compounds, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration. In some embodiments,a parental preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

EXAMPLES

The examples discussed below are intended to be purely exemplary of theinvention and should not be considered to limit the invention in anyway. The examples are not intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (for example, amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1 Materials and Methods Compounds

The following compounds were synthesized as described:

Compound 9.

A mixture of 2,3-dichloro-1,4-naphthoquin (11.35 g, 50 mmol) ando-aminophenol (5.45 g, 50 mmol), and anhydrous potassium acetate (9.8 g,100 mmol) in absolute ethanol (400 ml) was refluxed for 2 hours. Thesolvent was removed to 50 ml, and added cold water, filtered. The solidwas crystallization in acetone several times obtaining a yellow solid ascompound 9 that was matched with the literature report (Agarwal andSchafer, J. Org. Chem. 45: 2155-2161 (1980); Agarwal and Schafer, J.Org. Chem. 45: 5144-5149 (1980)).

Compound 9.E.

A mixture of 2,3-dichloro-1,4-naphthoquin (2.27 g, 10 mmol) ando-aminophenol (2.18 g, 20 mmol) in 50 ml of ethanol was heated at 60° C.for 0.5 h. The mixture was reduced to 20 ml and diluted with 500 ml ofwater, and the product was isolated by filtration and dried. The solidwas crystallized several times obtaining a yellow solid as compound 9.Ethat matched with the literature report (Agarwal and Schafer, J. Org.Chem. 45: 2155-2161 (1980)).

Compound 9.F.

A mixture of 2,3-dichloro-1,4-naphthoquin (2.27 g, 10 mmol) ando-aminophenol (2.18 g, 20 mmol), and 2 ml concentrated HCl in 50 ml ofethanol was refluxed for 2 h. The mixture was reduced to 20 ml anddiluted with 500 ml of water, and the product was isolated by filtrationand dried. The solid was crystallized several times obtaining a yellowsolid as compound 9.F that matched with the literature report (Agarwaland Schafer, J. Org. Chem. 45: 2155-2161 (1980)).

Compound 9.G.

A mixture of 2,3-dichloro-1,4-naphthoquin (2.27 g, 10 mmol) ando-aminophenol (2.18 g, 20 mmol), and 1 ml concentrated HCl in 20 ml ofmethanol was refluxed on a steam bath for 2 h. The mixture was dilutedwith 500 ml of water, and the product was isolated by filtration anddried. The solid was crystallized several times obtaining a yellow solidas compound 9.G that matched with the literature report (Agarwal andSchafer, J. Org. Chem. 45: 2155-2161 (1980)).

The following compounds were obtained from commercial vendors: 9.A,Sigma-Aldrich, St Louis, Mo., USA (cat. no. 255246); 9.B, Sigma-Aldrich(cat. no. 19123); 9.C, Ryan Scientific, Mt. Pleasant, S.C., USA (cat.no. RDR 00966); 9.D, ChemBridge, San Diego, Calif., USA (cat. no.5195211); 9.H, Princeton Biomolecular, Princeton, N.J., USA (cat. no.OSSL131788); 9.1, Sigma-Aldrich (cat. no. 424455); 9.J, Ryan Scientific(cat. no. AE-848/01277001); 9.K, Milestone Pharmatech, New Brunswick,N.J., USA (cat. no. 1526735); 9.L, Princeton Biomolecular (cat. no.OSSK_(—)460611); 9.M, Sigma-Aldrich (cat. no. Q1603); 19.A, RyanScientific (cat. no. STOCK3S-12829); 19.B, Sigma-Aldrich (cat. no.R750794); 19.C, Princeton Biomolecular (cat. no. OSSL_(—)038866).

Antibodies and Western Blot Analysis

Western blotting was performed on a 4-12% Bis-Tris SDS-PAGE system(NuPAGE; Invitrogen, Carlsbad, Calif., USA) as described previously(Costantini et al., 2006; Jonas et al., 2008; Jonas et al., 2010; Ko andPuglielli, 2007; Ko and Puglielli, 2009; Pehar et al., 2010). Thefollowing antibodies were used in this study: anti-acetylated lysine(monoclonal; cat. no. ab409; Abcam, Cambridge, Mass., USA); anti-BACE1(polyclonal; cat. no. ab2077; Abcam); anti-Myc (monoclonal; cat. no.sc-40; santa cruz, Santa Cruz, Calif., USA); anti-ATases/NAT8(polyclonal; cat. no. AP4957c; Abgent, San Diego, Calif., USA);anti-actin (monoclonal; cat. no. A1978; Sigma, St Louis, Mo., USA);anti-C99 (monoclonal; cat. no. M066-3; MBL, Woburn, Mass., USA);anti-acetylated H3 (polyclonal; cat. no. 06-599, Millipore, Billerica,Mass.); anti-acetylated H4 (polyclonal; cat. no. 06-866, Millipore,Billerica, Mass.); anti-aPCNA (polyclonal; cat. no. AP2835b, Abgent, SanDiego, Calif., USA).

Samples were imaged with classical chemiluminescence and with the LiCorOdyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, Nebr.,USA). For chemiluminescent detection, HRP-conjugated anti-mouse oranti-rabbit secondary antibodies were used at 1:6000 dilution in 3%BSA/TBST (GE Healthcare). Detection was performed with either Lumiglo(KPL) or ECL Plus (GE Healthcare). For infrared imaging, goatanti-rabbit Alexa Fluor 680-conjugated secondary antibodies were used.For quantification, values were normalized to the appropriate loadingcontrol (shown in the figures).

Real Time PCR

Total RNA was isolated using RNeasy Plus Mini Kit (Qiagen, Valencia,Calif., USA). One μg of total RNA was randomly reverse transcribed usingSuperScript III reverse transcriptase (Invitrogen, Carlsbad, Calif.,USA) according to the manufacturer's instructions. Quantitative PCRswere carried out in a LightCycler480 Real-time PCR System (Roche,Indianapolis, Ind., USA) using LightCycler480 SYBR Green I Master Mix(Roche). The cycling parameters were as follows: 95° C., 10 seconds; 55°C., 10 seconds; 72° C., 15 seconds, for a maximum of 40 cycles.

Controls without reverse transcription were included in each assay. PCRprimers specific to each gene were as follows:

ATase1/NAT8B-human, forward (SEQ ID NO: 1) 5′-CGATTACTGAAGCTGCCTCGA-3′,reverse (SEQ ID NO: 2) 5′-GGTTTTTTGGCAAGGAACCAC-3′; ATase2/NAT8-human,forward (SEQ ID NO: 3) 5′-TCCTTGCCAAAAAACCCTGG-3′, reverse(SEQ ID NO: 4) 5′-ATGCCCACCACCTTCTCTTCA-3′.

ATase1 and ATase2 expression levels were normalized against GAPDH levelsand are expressed as percent of control. PCR primers specific to GAPDHwere as follows:

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-human, forward(SEQ ID NO: 5) 5′-GAAGGTGAAGGTCGGAGTC-3′, reverse (SEQ ID NO: 6)5′-GAAGATGGTGATGGGATTTC-3′;

AcetylCoA:lysine acetyltransferase Assay

The acetyl:CoA lysine acetyltransferase activity of ATase1 and ATase2was assayed as described before (Ko and Puglielli, 2009). Briefly, weadapted a commercially available fluorescent kit (cat. no. 10006515,Cayman Chemicals) as follows: as source of the enzymatic activity, weused affinity purified ATase1-myc and ATase2-myc at the finalconcentration of 300 ng/μl; as donor of the acetyl group, we usedacetyl-CoA at the final concentration of 12.5 μM. ATase1 and ATase2 werepurified with the ProFound c-Myc-Tag IP/Co-IP Kit (Pierce) as suggestedby the manufacturer and already described in our previous work (Ko andPuglielli, 2009). The acetyltransferase assay was performed asrecommended by the manufacturer.

For kinetic analysis of ATase1 and ATase2 inhibition, the assays wereperformed in the presence of the indicated concentrations of compound 9,19 and 9.I. The concentration of acetyl-CoA was varied from 0.5 to 15mM. Values were plotted as a Lineweaver-Burk plot using Graphpad Prismsoftware.

Aβ Determination

For Aβ determinations in the conditioned media, H4 cells were plated in6-well Petri dishes. When 80-90% confluent, cells were washed in PBS andincubated in 1 ml of fresh medium for 48 hr. Secreted Aβ was determinedby standard sandwich ELISA as described before (Costantini et al., 2007;Costantini et al., 2006; Jonas et al., 2008; Jonas et al., 2010; Ko andPuglielli, 2009; Pehar et al., 2010). For each sample, the levels ofAβ40, Aβ42, and Aβtotal were quantified as triplicate based uponstandard curves run (on every ELISA plate) and then expressed as pmolAβ/mg of protein. Aβ42 was constantly found to be ˜25% of total Aβvalues.

Preparation of Cytosolic and Nuclear Fractions

Cytosolic and nuclear extracts were prepared as described before (Ko andPuglielli, 2007). For cytosolic extracts, cells were homogenized inhomogenization buffer containing 25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA,0.5 mM EGTA, and a protease inhibitor mixture. The homogenates werecentrifuged at 14,000×g for 15 min, and supernatants were collected ascytosolic proteins.

For nuclear extracts cells were scraped into ice-cold phosphate-bufferedsaline and collected by centrifugation. The cell pellets were suspendedin 3 volumes of lysis buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA,pH 8.0, 0.2% Nonidet P-40, 10% glycerol, and a protease inhibitormixture) followed by incubation on ice for 10 min. Cell suspensions weregently pipetted up and down; the lysates were then centrifuged at14,000×g for 5 min at 4° C. to obtain nuclear pellets. Nuclear pelletswere washed twice with cell lysis buffer (lacking Nonidet P-40 andprotease inhibitor mixture) and then resuspended in 2 volumes of nuclearextract buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA, pH 8.0, 420mM NaCl, 20% glycerol, and a protease inhibitor mixture). The nucleiwere extracted by incubation at 4° C. for 30 min with gentle agitationfollowed by centrifugation at 14,000×g at 4° C. for 5 min; the resultantsupernatant fraction was used as a nuclear extract.

Statistical Analysis

Results are always expressed as mean±S.D. of the indicated number ofdeterminations. The data were analyzed by ANOVA and Student's t testcomparison, using GraphPad InStat3 software. Statistical significancewas reached at P>0.05.

Example 2 ATase1 and ATase2 are Expressed in Neurons and areUp-Regulated in Alzheimer's Disease Brains

To confirm the relevance of ATase1 and ATase2 for AD neuropathology, wefirst assessed whether they are expressed in the brain and in cellularsystems that are relevant for the study of the nervous system. FIG. 1Ashows that the commercially available antibody used in this study couldrecognize transgenic ATase1 and ATase2 in Chinese Hamster Ovary (CHO)cells over-expressing myc-tagged versions of the human proteins. Neitheracetyltransferase could be significantly detected in non-transfected CHOcells. We next assessed the expression levels of the endogenousacetyltransferases in human neuroglioma (H4), human neuroblastoma(SH-SY5Y and SHEP) and rat pheochromocytoma (PC-12) cells as well asmouse primary neurons and mouse cerebral cortex. Both ATase1 and ATase2were identified, albeit with different intensity (FIG. 1B).Specifically, ATase1 appeared to be equally expressed while ATase2appeared to be more predominant in H4, SH-SY5Y and SHEP cells. Detectionof ATase2 in PC-12 cells required longer exposure (FIG. 1B; rightpanel). Assessment of a total extract of mouse cerebral cortex showedsignificant expression of ATase1 but very low expression of ATase2 (FIG.1B; lane 8). However, both were easily detectable in mouse primaryneurons suggesting a preferential expression of the ATases in neurons(FIG. 1B).

Interestingly, the expression levels of ATase2 increased with the age ofthe primary neurons (FIG. 1B; compare lane 6 and 7). Both we and othergroups have shown that, when in culture, primary neurons display aninternal timing that mimics cellular senescence (Costantini, 2006;Shen,2006). In particular, we reported that the levels of endogenous ceramideincrease with the age of the culture (Costantini, 2006). We alsoreported that ceramide treatment increases the mRNA levels of bothATase1 and ATase2 (Ko and Puglielli, 2009). Therefore, the activation ofATase2 in “older” neurons is consistent with our previous findings. Wealso noticed that ATase2 is more tightly regulated by ceramide thanATase1 (FIG. 5). Therefore, the above results indicate that bothacetyltransferases are expressed in the brain and neurons as well as incell lines that are commonly used for the study of the nervous system.They also suggest that in contrast to ATase1, which is constitutivelyexpressed, ATase2 acts as a regulated form of acetylCoA:lysineacetyltransferase.

Direct assessment of ATase1 and ATase2 mRNA levels in the brain oflate-onset AD patients revealed a significant increase, when compared toage-matched controls (FIG. 1C). Although these results might indicatedisease-relevant functions, they do not prove a direct cause-effect typeof relationship between ATase1/ATase2 levels and AD neuropathology. Infact, results obtained with postmortem AD tissue might be influenced bythe long duration of the disease, which could affect the genetic profileof the tissue. However, they are consistent with previous work showingthat AD patients also have a 3-fold increase in the levels of ceramidein brain areas that are affected by AD neuropathology ((Han,2002;Cutler, 2004); reviewed in (Puglielli, 2008)). We have alreadyreported that the levels of both ATases are tightly regulated by thelipid second messenger ceramide (FIG. 5; see also (Ko and Puglielli,2009)).

Example 3 ATase1 and ATase2 Inhibitors Down-Regulate Both Levels andActivity of BACE1

We previously described a fluorescent assay that is able to assess theacetylCoA:lysine acetyltransferase activity of ATase1 and ATase2 invitro (Ko and Puglielli, 2009). The assay employs affinity purifiedATase1 or ATase2 (as enzymes), acetyl-CoA (as donor of the acetyl group)and a recombinant peptide corresponding to the N-terminal tail of thehistone protein H3 (as acceptor of the acetyl group). The H3 peptide waspreferred to affinity purified BACE1 because it is acetylated in aconformation independent manner ((Ko and Puglielli, 2009) and data notshown) and, therefore, more suitable for large screening approaches. Toidentify possible ATase1 and ATase2 inhibitors we used the assay toscreen a single library of 14,400 small molecules/compounds. The screenidentified 186 small molecules/compounds able to inhibit ATase1, ATase2or both in vitro. Importantly, 93 compounds were unique to our screensuggesting unique properties. Each compound was then screened forcytotoxic properties by using multi-plex cytotoxicity assays on avariety of cell lines, including human neuroblastoma and humanneuroglioma. Of the above 186 small molecules/compounds, only 30 did notcause significant cytotoxicity when used at 10 μM final concentration. Asummary of the screen is shown in FIG. 6. The above 30 compounds wereindividually screened on human neuroglioma (H4) cells for their abilityto reduce the levels of BACE1 and the generation of Aβ. Two compounds, 9and 19, produced significant changes and were object of further study.

The compounds display very different chemical and structural features(FIG. 2A), which probably explain the different kinetics of inhibitionfor acetyl-CoA, the donor of the acetyl group. In fact, compound 9displayed a competitive inhibition with ATase1 and a non-competitiveinhibition with ATase2 while compound 19 displayed non-competitiveinhibition with both enzymes (FIG. 2B,C). The IC50 for compound 9 was˜3.9 μM for ATase1 and ˜0.79 μM for ATase2; the IC50 for compound 19 was˜13.3 μM for ATase1 and 18.2 μM for ATase2. The fact that compound 9uses two different mechanisms of reversible inhibition with ATase1 andATase2 is consistent with our early report showing that, although highlyidentical, the two enzymes display a few biochemical differences (Ko andPuglielli, 2009).

To assess their biological effects, we treated human neuroglioma (H4)cells with 10 μM of both compounds. In both cases, we observed asignificant reduction in the levels of BACE1 (FIGS. 3A-D), althoughcompound 9 appeared to act as a more potent inhibitor. This mightreflect different kinetics of inhibition as well as different IC50s (seeFIG. 2). Alternatively, it might also be explained by differentcell-permeability properties. In fact, the cLog P for compound 9 and 19is 5.03 and 3.19, respectively. The cLog P (or partition coefficient) isan indication of partition properties of a compound between hydrophobicand hydrophilic compartments. Higher values indicate preferentialdistribution in hydrophobic environments and, as such, increasedpermeability across biological membranes.

Interestingly, both compounds preferentially affected the mature form ofBACE1 (FIG. 3A,C). We previously reported that the ER-based acetylationof nascent BACE1 prevalently affects the levels of mature BACE1(Costantini, 2007;Ko and Puglielli, 2009). In fact, non-acetylatedmutants of BACE1 are correctly synthesized in the ER but rapidlydegraded in the ERGIC before they can complete maturation (Costantini,2007;Jonas, 2008). The decreased levels of BACE1 were paralleled bydecreased levels of Aβ in the conditioned media (FIG. 3E) and decreasedcellular levels of C99 (FIG. 7). As mentioned above, C99 is theimmediate product of BACE1-mediated cleavage of APP, whereas Aβrepresents one of the final products of the sequential processing of APPby BACE1 and γ-secretase (Puglielli, 2008). Parallel changes in C99 andAβ directly reflect the steady-state levels and activity of BACE1. Takentogether, the above results indicate that both compound 9 and 19 affectthe metabolism of BACE1 and the rate of Aβ generation.

Lysine acetylation was initially discovered as a covalent modificationof nuclear and cytosolic proteins, which included histones, differentfamilies of transcription factors and cytoskeleton-associated proteins(Yang and Seto, 2007). Therefore, it is possible that, although screenedfor their ability to inhibit ATase1 and ATase2 in vitro, compound 9 and19 might interfere with the acetylation of several other classes ofproteins. To address this point we decided to assess the acetylationprofile of both cytosolic and nuclear proteins. Neither compound 9 norcompound 19 produced significant changes (FIGS. 8A,B). However, directassessment of the acetylation profile of the histone protein H3 and H4revealed a slight reduction (FIG. 8C). This effect was more evident withhistone H4 then H3. When taken together, the results displayed in FIG. 8suggest that compound 9 and compound 19 are highly selective. In fact,the most evident decrease in lysine acetylation was observed withhistone H4 suggesting that they only interfere with theacetyl-CoA:lysine acetyltransferase activity of a very limited number ofnon-ER acetyltransferases, perhaps specific to histone H4. It is alsoworth stressing that neither compound inhibited the in vitro activity ofthe histone acetyltransferase p300/CBP (data not shown).

Next we decided to analyze structure-activity properties of bothcompounds. In the case of compound 9, we designed 13 different chemicalderivatives and assessed IC50s in vitro. The results displayed in Table3 show that some modifications improved while others abolished theinhibitory properties of the parent compound.

TABLE 3 IC_(50s) of compound 9 derivatives

Although the trend was overall similar, we observed some degree ofdifference when comparing IC50s for ATase1 and ATase2, suggesting thatcertain structural determinants affect the two enzymes differently(Table 3). The progressive loss of activity observed with derivatives9.E-9.G is particularly interesting because it highlights an importantstructural feature (FIG. 9). In fact, compound 9.E is a rearrangedacetal in which the central ring junction is pyramidalized by thepresence of a hydroxyl group. This compound is bent slightly relative tothe planar compound 9 (FIG. 9; pseudo-top view) and shows decreasedactivity (Table 3). Replacement of this hydroxyl group with methoxyl orethoxyl group obliterates activity (Table 3); these modifications alsoappear to cause a more pronounced bending of the molecule based onmodeling (FIG. 9; pseudo-top view), and suggest that the planarstructure of compound 9 is essential for its inhibitory activity. Incontrast, the modifications introduced in derivatives 9.A-9.D revealedareas that can be modified without affecting the enzymatic properties(FIG. 10; marked in yellow). The region highlighted in the “left” ringis particularly interesting because easily accessible and can befunctionalized for a variety of purposes, such as increasing solubility,attachment to cell-penetrating peptides, or conjugation to biomolecules.The highest activity was observed for compound 9.0 in which the centralring oxygen was replaced with nitrogen and the carbonyl-containing ringwas tautomerized to a phenol, suggesting that these modifications can bebeneficial. However, shrinking the carbonyl-containing ring to a5-membered ring obliterated the activity (compound 9.H of Table 3; seealso FIG. 11) indicating that the 6-membered ring is critical. Finally,reduction of the structure of the parent compound to three or two ringsresulted in a complete loss of activity (FIG. 11). The only exceptionwas derivative 9.1, which retained inhibitory properties (Table 3; seealso FIG. 11). The only significant difference between 9.I and 9.J wasin one hydroxyl and one oxygen group, which are missing in 9.J (FIG.11).

The results obtained with the derivatives of compound 19 were much moredramatic. In fact, removal of the right side-chain (see FIG. 12;highlighted) reduced or completely abolished the activity (Table 4).Reduction of the structure to a two ring unit also abolished activity(data not shown). Therefore, overall the activity of compound 19 istightly linked to the side chain on the fourth ring and does not offerroom for modifications that could be used to improve biologicalproperties.

TABLE 4 IC_(50s) of compound 19 derivatives

In conclusion, when taken together, the above results clearly indicatethat the inhibitory properties of both compound 9 and 19 are tightlydependent on their structure. This conclusion probably explains the highdegree of selectivity that we observed in cellular systems (see FIG. 3and FIG. 8).

Example 4 Compound 9 Causes Degradation of the ATases

To assess whether the chemical modifications introduced in compound 9also affected the biological properties, we tested the most activederivatives in cellular systems. H4 cells were treated with 10 μM ofcompound 9, 9.A, 9.B, 9.C, 9.E and 9.I. The results show that only 9.Iretained significant ability to affect the levels of BACE1 (FIGS. 4A,B)and the generation of Aβ (FIG. 4C). Surprisingly, these results appearedto correlate with the endogenous levels of ATase1 and ATase2 (FIGS.4A,D,E). In fact, both compound 9 and 9.I were able to significantlydown-regulate the acetyltransferases (FIGS. 4A,D,E). Compound 9, whichwas more active in down-regulating BACE1 levels, also produced a moredramatic decrease in the levels of the ATases (FIGS. 4A,D,E). Finally,compound 9.I appeared to affect ATase1 more dramatically than ATase2(FIGS. 4A,D,E). The close relationship between levels of the ATases andBACE1 was dose dependent. In fact, increasing concentrations of compound9 caused a dose-dependent decrease in the levels of both the ATases andBACE1 (FIG. 13). This behavior is consistent with our previous resultswhere we over-expressed or down-regulated the ATases (Ko and Puglielli,2009). Since, the above experiments might potentially be affected by ourability to detect endogenous ATase1 and ATase2, we also treated CHOcells over-expressing transgenic ATase1 or ATase2. Western blotassessment of ATase1 and ATase2 levels confirmed that compound 9significantly decreases the levels of both proteins.

Example 5 Compound 9 Crosses the Blood Brain Barrier and ShowsBeneficial Effects in AD Mouse Model Compound 9 Crosses the Blood BrainBarrier

To assess BBB permeability properties, a group of 5 wild-type micereceived 50 mg/kg/day of compound 9 for 1 week. Animals were thensacrificed and CSF collected for biochemical analysis. Treatment waslimited to 1 week because this length of time is usually sufficient toreach equilibrium in biological fluids (Ito, et al., 1998; Singh, 2006;Houston and Galetin, 2008). Concentration and duration of the treatmentwas based on previous studies with drug-like compounds having similarmass and solubility properties (Ito, et al., 1998; Singh, 2006; Houstonand Galetin, 2008).

Mass spectrometric assessment of the CSF was able to detect compound 9in all treated animals, confirming our earlier predictions (FIG. 14).

Compound 9 Prevents LTP Defects and Reduces BACE1 and Aβ Levels in MouseAD Model

In order to assess disease-relevant functions, we performed a pilotstudy with APP_(695/swe) mice, a commonly-used mouse model of AD.APP_(695/swe) mice express a modified mouse APP cDNA encoding the695-amino acid isoform with a “humanized” Aβ domain that includes thefamilial AD-associated Swedish double mutation. APP_(695/swe) micedevelop synaptic deficits at 3-5 months of age, well before any sign ofneurodegeneration can be observed. The cognitive deficits as well as theamyloid plaques can be detected only later, at approximately 12-18months of age (reviewed in Duyckaerts, et al., 2008).

A group of non-transgenic and APP_(695/swe) mice were fed a dietcontaining 50 mg/kg/day of compound 9. Treatment started at weaning (˜6weeks of age) and lasted 4 months. At the end of treatment, the animalswere sacrificed and the electrophysiological properties of hippocampalbrain slices (as induced by theta burst stimulation to the Schaffercollaterals) were studied.

As described in our previous work (Pehar, et al., 2010), APP_(695/swe)mice displayed a marked defect in the late component of the long-termpotentiation (LTP) (FIG. 15A) in the absence of significant deficits inthe presynaptic component of synaptic transmission (data not shown; seePehar, et al., 2010). However, these changes were drastically preventedby compound 9. Importantly, no effect was observed in non-transgenicmice (FIG. 15B), indicating that the compound acts by preventingdisease-relevant events rather than by improving intrinsic synapticproperties.

The electrophysiological changes were also accompanied by a markedreduction in BACE1 levels (FIG. 16) and a ˜50% reduction in soluble Aβin the brain homogenates of APP_(695/swe) transgenic mice (Table 5).Note that at this age, the animals have not yet developed plaques. Asexpected from our previous results in cellular models (Ko and Puglielli,2009), no effect on the Aβ₄₂/Aβ_(total) ratio was observed (data notshown). In fact compound 9 affects BACE1 levels and, as such, impacts onthe generation of all Aβ species.

TABLE 5 Aβ levels in brain homogenates (cortex) of APP_(695/swe) mice.Aβ (ng/gr) Control diet 335 ± 41 Compound 9 178 ± 14 P < 0.005

The foregoing description is considered as illustrative only and is notintended to limit the claimed invention. Numerous modifications andchanges may readily occur to those skilled in the art. The invention isnot limited to the exact construction and operation shown and described,and accordingly, all suitable modifications and equivalents areconsidered to fall within the scope of the invention.

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1. A pharmaceutical composition for treating Alzheimer's diseasecomprising: (a) an effective amount of a compound selected from thecompounds of Formula I:

wherein: R₁, R₂, R₃, R₄, and R₂ are each independently selected fromhydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, andC₁-C₁₀ alkyl, wherein R₉ and R₁₀ are independently selected from C₁-C₅alkyl; R₈ is selected from hydrogen, halogen, —OH, —NH₂, and —SH, or isabsent; R₅ and R₆ are each independently selected from hydrogen,halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₁₀ alkyl,wherein R₉ and R₁₀ are independently selected from C₁-C₅ alkyl; or R₅and R₆ together are

wherein R₁₁, R₁₂, R₁₃, and R₁₄ are each independently selected fromhydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, andC₁-C₁₀ alkyl, wherein R₉ and R₁₀ are independently selected from C₁-C₅alkyl; X₁ is selected from N, NH, O, and S; X₂ is selected from N, O,and S; X₃ is selected from —OH, ═O, —NH₂, ═NH, —SH, and ═S; and whereinthe X₁ to carbon a (C_(a)) bond and the carbon b (C_(b)) to carbon c(C_(c)) bond are both double bonds, or the C_(a) to C_(b) bond is adouble bond; or pharmaceutically acceptable salts thereof; and (b) apharmaceutically acceptable carrier.
 2. The pharmaceutical compositionof claim 1, wherein R₁, R₃, R₄, and R₂ are each independently selectedfrom the group consisting of hydrogen and halogen.
 3. The pharmaceuticalcomposition of claim 2, wherein R₁, R₃, and R₄ are each hydrogen and R₂is halogen.
 4. The pharmaceutical composition of claim 1, wherein R₅ andR₆ are each independently selected from the group consisting ofhydrogen, halogen, and methyl; or wherein R₅ and R₆ together are

wherein R₁₁, R₁₂, R₁₃, and R₁₄ are each independently selected from thegroup consisting of hydrogen and halogen.
 5. The pharmaceuticalcomposition of claim 4, wherein R₅ and R₆ are each hydrogen or halogen;or wherein R₅ and R₆ together are


6. The pharmaceutical composition of claim 1, wherein R₂ is selectedfrom the group consisting of hydrogen, halogen, —OH, —OR₉, —SH, —SR₉,—NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₆ alkyl, wherein R₉ and R₁₀ areindependently selected from any C₁-C₃ alkyl.
 7. The pharmaceuticalcomposition of claim 1, wherein R₈ is selected from the group consistingof hydrogen, halogen, —OH, and —SH, or wherein R₈ is absent.
 8. Thepharmaceutical composition of claim 7, wherein X₂ is N and R₈ is absent.9. The pharmaceutical composition of claim 7, wherein X₂ is O and R₈ isselected from hydrogen, halogen, —OH, and —SH.
 10. The pharmaceuticalcomposition of claim 1, wherein the compound is selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.
 11. A pharmaceuticalcomposition for treating Alzheimer's disease comprising: (a) aneffective amount of a compound selected from the compounds of FormulaII:

wherein: R₁ is selected from the group consisting of hydrogen, halogen,—OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C₁₀ alkyl,wherein R₁₁ and R₁₂ are independently selected from C₁-C₆ alkyl; R₂ isselected from the group consisting of hydrogen, halogen, —OH, —SH, and—NH3; R₃ to R₁₀ are each independently selected from the groupconsisting of hydrogen, halogen, —OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁,—NR₁₁R₁₂, and C₁-C₁₀ alkyl, wherein R₁₁ and R₁₂ are independentlyselected from C₁-C₆ alkyl; X₁ is selected from the group consisting ofO, S, and NH; X₂ and X₃ are each independently selected from the groupconsisting of O, S, and NH; or pharmaceutically acceptable saltsthereof; and (b) a pharmaceutically acceptable carrier.
 12. Thepharmaceutical composition of claim 11, wherein R₃ to R₁₀ are eachindependently selected from the group consisting of hydrogen, halogen,—OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C₁₀ alkyl,wherein R₁₁ and R₁₂ are independently selected from C₁-C₃ alkyl.
 13. Thepharmaceutical composition of claim 11, wherein R₃ to R₁₀ are eachindependently selected from the group consisting of hydrogen andhalogen.
 14. The pharmaceutical composition of claim 11, wherein R₁ isselected from the group consisting of hydrogen, halogen, —OH, —OR₁₁,—SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C₁₀ alkyl, wherein R₁₁ andR₁₂ are independently selected from any C₁-C₄ alkyl.
 15. Thepharmaceutical composition of claim 14, wherein R₁ is selected from thegroup consisting of hydrogen, halogen, —OH, —OR₁₁, —SH, —SR₁₁, —NH₂,—NHR₁₁, —NR₁₁R₁₂, and C₁-C₁₀ alkyl, wherein R₁₁ and R₁₂ areindependently selected from any C₁-C₃ alkyl.
 16. The pharmaceuticalcomposition of claim 11, wherein R₂ is selected from the groupconsisting of hydrogen and halogen.
 17. The pharmaceutical compositionof claim 11, wherein X₁ is NH.
 18. The pharmaceutical composition ofclaim 11, wherein X₂ and X₃ are each O.
 19. The pharmaceuticalcomposition of claim 11, wherein the compound is selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.
 20. A method of treating,preventing, delaying the onset of, slowing the progression of, oralleviating at least one symptom of Alzeimer's disease comprisingadministering to a patient a pharmaceutical composition comprising aneffective amount of an inhibitor of ATase1 and/or ATase2, wherebyAlzheimer's disease in the patient is prevented, delayed in onset, orslowed in progression, or whereby at least one symptom of Alzheimer'sdisease is alleviated.
 21. The method of claim 20, wherein the inhibitorof ATase1 and/or ATase2 is a compound selected from the compounds ofFormula I:

wherein: R₁, R₂, R₃, R₄, and R₂ are each independently selected fromhydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, andC₁-C₁₀ alkyl, wherein R₉ and R₁₀ are independently selected from C₁-C₅alkyl; R₈ is selected from hydrogen, halogen, —OH, —NH₂, and —SH, or isabsent; R₅ and R₆ are each independently selected from hydrogen,halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, and C₁-C₁₀ alkyl,wherein R₉ and R₁₀ are independently selected from C₁-C₅ alkyl; or R₅and R₆ together are

wherein R₁₁, R₁₂, R₁₃, and R₁₄ are each independently selected fromhydrogen, halogen, —OH, —OR₉, —SH, —SR₉, —NH₂, —NHR₉, —NR₉R₁₀, andC₁-C₁₀ alkyl, wherein R₉ and R₁₀ are independently selected from C₁-C₅alkyl; X₁ is selected from N, NH, O, and S; X₂ is selected from N, O,and S; X₃ is selected from —OH, ═O, —NH₂, ═NH, —SH, and ═S; and whereinthe X₁ to carbon a (C_(a)) bond and the carbon b (C_(b)) to carbon c(C_(c)) bond are both double bonds, or the C_(a) to C_(b) bond is adouble bond; or pharmaceutically acceptable salts thereof.
 22. Themethod of claim 21, wherein the inhibitor of ATase1 and/or ATase2 isselected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 23. The method of claim20, wherein the inhibitor of ATase1 and/or ATase2 is a compound selectedfrom the compounds of Formula II:

wherein: R₁ is selected from the group consisting of hydrogen, halogen,—OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁, —NR₁₁R₁₂, and C₁-C₁₀ alkyl,wherein R₁₁ and R₁₂ are independently selected from C₁-C₆ alkyl; R₂ isselected from the group consisting of hydrogen, halogen, —OH, —SH, and—NH3; R₃ to R₁₀ are each independently selected from the groupconsisting of hydrogen, halogen, —OH, —OR₁₁, —SH, —SR₁₁, —NH₂, —NHR₁₁,—NR₁₁R₁₂, and C₁-C₁₀ alkyl, wherein R₁₁ and R₁₂ are independentlyselected from C₁-C₆ alkyl; X₁ is selected from the group consisting ofO, S, and NH; X₂ and X₃ are each independently selected from the groupconsisting of O, S, and NH; or pharmaceutically acceptable saltsthereof.
 24. The method of claim 23, wherein the inhibitor of ATase1and/or ATase2 is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.